The topic of the original funded grant proposal was phosphoinositide regulation of the heat- and capsaicin sensitive TRPV1, and the cold- and menthol-sensitive TRPM8 channels. The current renewal proposal continues to study phosphoinositide regulation of TRPV1, and addresses an important unsolved problem, brought to light by a recent higher resolution structure in lipid nanodiscs, which found that the capsaicin/vanilloid binding site is occupied by a phosphoinositide, and proposed that it stabilizes the channel in the resting state, and vanilloids activate TRPV1 by replacing the lipid. PI(4,5)P2 and PI(4)P however are well established positive cofactors/regulators of TRPV1, which is difficult to reconcile with this model. The exact nature of the phosphoinositide lipid, however, is not well resolved in the structure. In Aim1 we will elucidate the nature of the phosphoinositide in the vanilloid binding site, using the combination of computational modeling, site directed mutagenesis, whole cell and excised patch electrophysiology and planar lipid bilayers. The TRPM3 ion channel is expressed in Dorsal Root Ganglion (DRG) neurons; its genetic deletion in mice results in altered sensitivity to noxious heat. TRPM3 is activated by heat, and chemical agonists, such as Pregnenolone Sulphate (PregS) and CIM0216. We found that this channel requires phosphoinositides for activity, and we also found that agonists of phospholipase C (PLC)-coupled receptors inhibit TRPM3. This inhibition, however, was not alleviated by intracellular delivery of excess PI(4,5)P2, and was reduced by a protein that binds the ?? subunits of heterotrimeric G-proteins (G?? sink). This finding points to the dominance of G?? signaling over PLC activation in regulating TRPM3. Activation of Gi-coupled receptors that do not activate PLC also robustly inhibited TRPM3 activity, and the effect was reduced by G?? sinks. Co-expression of G?? in intact cells, and application of purified G?? to excised inside-out patches also inhibited TRPM3, and we detected biochemical interaction between TRPM3 and G? by co-immunoprecipitation. These data suggest that G?? subunits are direct negative regulators of TRPM3. We also found that activation of endogenous Gi- coupled GABAB and opioid receptors inhibited PregS-induced Ca2+ signals in DRG neurons. In Aims 2 and 3, we will test predictions of our model of TRPM3 regulation, and elucidate the molecular determinants of this effect using a combination of molecular biology, patch clamp, planar lipid bilayer, skin-nerve electrophysiology, fluorescence-based cellular imaging techniques, and animal behavior.